The invention pertains to methods of determining the presence of periodontal disease in mammals, and more particularly to improved methods of determining active periodontal disease by assaying for the presence of suprathreshold levels of the enzyme aspartate aminotransferase in oral fluid samples.
Periodontal diseases are inflammatory diseases of microbial etiology affecting the supporting tissues of the teeth. These diseases, which affect over 70% of the adult population, are the leading causes of tooth loss in people over 35 years of age. Costs associated with periodontal disease, including cost of treatment and the economic cost due to loss of productivity, is extremely high. It was estimated in 1976 that the cost of effective management of all those suffering from periodontal disease (more than 100 million people) would be many times the 1.5 billion dollars spent at that time [see "Evaluation of NIDR Periodontal Disease Research Activity--Report of the Ad Hoc Scientific Evaluation Panel", National Institutes of Dental Research, Washington, D.C. (April 1976)].
The term "periodontal disease" encompasses two major subclasses of disease, gingivitis and periodontitis. "Gingivitis" is characterized by inflammation of the gums in the absence of bone and attachment loss. See, Loe, H. and P. Silness, Acta Odont. Scand. 21:533 (1963). "Periodontitis" is generally accepted to be an advanced stage of gingivitis, further characterized by formation of periodontal pockets between the gum tissue and tooth, followed by loss of bone from the tooth and weakening of tooth attachment, eventually leading to tooth loss. See, Ramfjord, S., J. Periodontal. 38:602 (1967). Periodontitis may be further classified, e.g., juvenile periodontitis, local periodontitis, acute necrotizing periodontitis, chronic inflammatory periodontitis (CIPD). CIPD is the most common form of periodontitis among American adults and is characterized by loss of attachment of periodontal ligament to cementum, apical migration of junctional epithelium, and loss of alveolar bone. Both gingivitis and periodontitis are characterized by accumulation of crevicular fluid (a transudate of serum) at the junction of the teeth and gums.
Although periodontal disease is one of the most prevalent bacterial diseases in the civilized world, its diagnosis has been based, until recently, primarily upon subjective observational indices sch as those of Loe and Silness, supra, for gingivitis and Ramfjord, supra, for periodontitis. These indices are based on criteria such as bleeding on gentle probing, pocket depth, attachment loss and radiographic evidence of bone loss. Unfortunately, these clinical indicators, with the exception of bleeding on probing (bleeding of gum tissue due to probing of the gum line or pocket with a hard instrument, e.g. probe or curet), are generally acknowledged to be reflective of prior damage resulting from past disease as opposed to active periodontal disease. Further, even the diagnostic value of bleeding on probing has been questioned. See, Haffajee, Socransky and Goodson, J. Perio. 10:257-265 (1963). Characteristics additionally hindering diagnosis of the disease arise from the fact that in its early stages it may be asymptomatic and that it is frequently episodic, with a cyclical pattern of destructive activity interspersed with periods of latency or spontaneous partial regression.
Recently, several novel methods for the diagnosis of periodontal disease have been developed. One method takes advantage of the fact that gingivitis and periodontitis are characterized by the accumulation of crevicular fluid at the junction of the teeth and gums. Measurement of a large volume of crevicular fluid between the teeth and gums can indicate the presence of periodontal disease. An instrument known as the Periotron (Harco Electronics Ltd.; Winnipeg, Canada) is used to galvanometrically measure the volume of crevicular fluid absorbed by small strips of porous material known as Periopaper (Harco; Tustin, Calif.) which are inserted into the crevicular space between the tooth and gum.
McNamara, U.S. Pat. No. 3,691,018 discloses a diagnostic method for the early detection of incipient disease wherein crevicular fluid is tested for the presence of .beta.-D-galactosidase. The patent discloses that a strip formed from a nylon Millipore filter having a pore size of one micron may be used to collect crevicular fluid. The fluid is then tested for the presence of .beta.-D-galactosidase at two different pH's (5.0 and 7.5) one indicative of mammalian produced enzyme and the other indicative of bacterial produced enzyme. The porous strips are incubated for approximately 2 hours at 37.degree. C. with suitable substrates in buffer solution at pH 5 and pH 7.5. The strips are then removed and post-coupled with a diazonium salt for one minute to produce a color reaction. The intensity of color reaction for the sample at each pH is judged according to a standard of (1) no color; (2) slight intensity; (3) moderate intensity or (4) deep intensity. Color intensities of samples incubated at each of the two pHs are determined and compared to a master table in order to determine the presence and severity of periodontal disease.
Recently it has been disclosed, however, that the presence of elevated levels of the enzyme aspartate aminotransferase (AST) in crevicular fluid is highly correlative of the presence of active periodontal disease. Chambers, EPO patent application Publication No. 151,536 published Aug. 14, 1985. The presence of elevated levels of this enzyme in crevicular fluid is also disclosed to be predictive of a high probability of progressive, as opposed to non-progressive, periodontal disease and corresponding tissue damage. Increased levels of blood serum AST have previously been correlated with a wide variety of other conditions including acute myocardial infarction, pulmonary embolism, acute pancreatitis, viral and toxic hepatitis, and acute cirrhosis.
The enzyme aspartate aminotransferase [EC 2.6.1.1; L-aspartate:2-oxoglutarate aminotransferase] (previously known as glutamic aspartic transaminase, glutamic aspartic aminotransferase, glutamic aspartic aminopherase, glutamic oxaloacetic transaminase, GOT, G. O. T., or GO-T) (hereinafter referred to as AST) catalyzes the reaction: ##STR1## Pyridoxal phosphate is required as a prosthetic group. The enzyme also catalyzes other reactions (e.g., betasulphinyl pyruvate to L-cysteinesulphonate and betasulphonyl pyruvate to L-cysteate) but at a much slower rate.
AST is found in both the mitochondria and cytoplasm of eukaryotic cells. Both forms have a molecular weight of about 90,000 daltons and consist of 2 approximately equal size subunits, but differ in their physical and chemical characteristics and amino acid composition. AST is involved in a variety of catabolic and anabolic pathways for amino acids. See Lehninger, A. L., Biochemistry, 2nd ed. (Worth Publishers, New York, 1975).
According to the method of Chambers, crevicular fluid is collected from the interface of the gum and tooth by means such as a microsyringe, capillary tube or absorbant strip. The volume of material is measured and the presence of AST in the collected sample of crevicular fluid is determined by either colorimetric or immunological assay.
A wide variety of colorimetric assays are known for the detection of AST in serum. These assays actually analyze for the presence of oxaloacetate formed from the AST catalyzed reaction of aspartate and alpha-ketoglutarate. In one procedure, the production of oxaloacetate by Reaction I, supra, is coupled with the formation of a 2,4-dinitrophenyl-hydrazone-derivative which has a reddish-brown color and absorbs light at 520 nm: ##STR2##
In these procedures, the serum sample is incubated with excess amounts of L-aspartic acid and alphaoxoglutaric acid and excess 2,4-dinitrophenylhydrazone is then added. The reaction mixture is then further incubated to allow for conversion of any oxalacetate to its 2,4-dinitrophenylhydrazone derivative the color of which is brought out by addition of excess alkali. Chambers, supra, discloses use of this technique for the detection of AST present in crevicular fluid.
There are several disadvantages to procedures utilizing 2,4-dinitrophenylhydrazone, however. Incubation times are long and oxaloacetate accumulates over the course of the enzyme reaction resulting in inhibition of both isoenzymes. Further, mitochondrial AST is more sensitive to product inhibition and tends to be underestimated by this procedure.
In an alternative procedure known for use in detection of serum AST and disclosed by Chambers, supra, for detection of AST in crevicular fluid, oxaloacetate produced by reaction I is converted to pyruvate which is subsequently converted to the pyruvate-2,4-dinitrophenylhydrazone derivative. The excess aniline citrate is added after incubation of the sample with the substrate and before addition of the 2,4-dinitrophenylhydrazone reagent. After addition of the 2,4-dinitrohydrazone, the reaction mixture is incubated to allow for conversion of the pyruvate to its dinitrophenylhydrazone derivative which is brought out by the addition of excess alkali. ##STR3##
Examples of commercially available AST assays using this procedure are A-gent.TM. Aspartate Aminotransferase Assay (Abbott Cat. No. ABA-50, ABA-100, Abbott-VP, Abbott Laboratories, Chicago, Ill.), and Worthington Statzyme.RTM. GOT (Worthington Cat. No. CGOT, Worthington Diagnostic Systems, Inc., Freehold, N.J.).
Also known for the detection of AST are methods utilizing azozene dyes for reaction with oxaloacetate. These methods are significantly more rapid than those utilizing 2,4-dinitrophenyl hydrazone, do not cause product inhibition of AST and avoid use of alkali in developing the color reaction. Forgione, U.S. Pat. No. 3,875,014 discloses test indicators for the determination of AST concentrations in sera utilizing the pair of reactions ##STR4##
The test indicator of Forgione, comprises a pair of bibulous materials, adhered to each other with an adhesive which is selectively permeable to oxaloacetic acid, the first of which comprises the substrates L-aspartic acid and .alpha.-ketoglutaric acid. The second comprises a dried diazonium salt. The indicator is contacted with sera which, if it contains AST, catalyzes the reaction of the substrates to form oxaloacetic acid. Oxaloacetic acid then diffuses to the second strip and activates a color reaction with a diazonium salt.
Methods and various materials for the detection of AST utilizing diazonium salts are disclosed in Rej, "Measurement of Aminotransferases: Part 1. Aspartate Aminotransferase", CRC Critical Reviews in Clinical Laboratory Sciences, Vol. 21, No. 2, pp. 98-186 (1984). This reference discloses suitable diazonium salts including, Fast Violet B, {4-amino-2.5-diethoxy benzanilide (6-benzamido-4-methoxy-5-toluidine) diazonium chloride}; Fast Red PDC/Ponceau L, {N'-butyl-4-methoxymetanilamide diazonium salt}; Fast Red KL, {2-amino-4-methoxybenzamide diazonium salt}; Fast Scarlet GG, 2.5-dichloroaniline diazonium salt]; Fast Red RC, {5-chloro-2-methoxyaniline-1-diazonium chloride}; Fast Blue BB, {4-amino-2.5-diethoxybenzanilide diazonium chloride.}; Fast Blue B, {3.3'-dimethoxydiphenyl-4,4'-tetrazonium chloride} and Fast Blue RR, {4-amino-2.5-dimethoxybenzanilidine diazonium chloride}. Use of diazonium salts for detection of AST as disclosed in Forgione and Rej require either the subjective evaluation of color intensities as compared against a color chart or automated procedures which are only semi-quantitive and suffer from difficulties in calibration.
Because of the problems associated with techniques utilizing 2,4-dinitrohydrazone or diazonium dyes, a preferred alternative procedure is known wherein oxaloacetate produced by AST catalyzed reaction of L-aspartate and alpha-ketoglutarate is converted to malate in a second reaction by malate dehydrogenase with the cofactor nicotine adenine dinucleotide (NAD), in the presence of its reduced form, nicotine adenine dinucleotide (NADH). See, Hochstrasser, U.S. Pat. No. 4,059,407. NADH absorbs ultraviolet light at 340 nm, thus the rate of conversion of oxaloacetate to malate can be followed by monitoring the rate of disappearance of NADH at 340 nm. While the NADH system has the disadvantage of requiring a spectrophotometer (because NADH absorbs light in the ultraviolet spectrum) it has advantages of reproducibility and of quantitativeness as oxaloacetate is removed from the reaction mixture during the course of reaction so as to prevent product inhibition. Because of its numerous advantages, the system is considered the most durable and is currently the basis for national and international efforts for standardization of AST measurements. Rej, supra. The diazonium salt AST detection systems have largely been superseded by the NADH system in all areas with the exception of specialized (nonquantitative) electrophoretic applications. Rej, supra, pp. 139-141.
Desired by the art are techniques whereby the presence of a compound in excess of a predetermined concentration may be detected by a simple yes-no test not requiring a color chart. Opp, U.S. Pat. No. 4,471,055 discloses a process and a kit for the detection of aldehyde concentrations in a sample in excess of a predetermined concentration. Two reaction systems are employed. The first reaction system acts in transforming quantitatively to a first reaction product the amount of aldehyde equal to the predetermined concentration. The second reaction system then acts to transform any remaining aldehyde to a second reaction product which is visibly detectable.
Hochstrasser, supra, discloses disposable chemical indicators for the measurement of concentrations of a wide variety of materials in biological fluids including ketones, proteins and AST. The indicator registers the concentration of substance detected with multiple indicia which read either "on" or "off" at specific threshold values and thus eliminate the need for subjective judgment of color intensity and the need for color charts and the like. Reagents are used which give a visual indication when exposed to specific concentrations of the substance to be tested. The patent discloses reagent systems which comprise a fixed amount of a titrant which reacts with one of the products in a reduction involving the material being analyzed and proceeding to completion with the formation of one or more products, one of which reacts quantitatively with the titrant, and is thus not permitted to accumulate (and produce a color signal) until all of the titrant is consumed at which time the accumulation of the product becomes visible to the eye or to a spectrophotometric device.
Hochstrasser, utilizes the NADH/malic acid , reaction system for the detection of AST with indicator systems disclosed as follows: ##STR5##
Aspartic acid, alpha-ketoglutaric acid, malic acid dehydrogenase and NADH are identified by Hochstrasser as "reagents". Suitable indicators are disclosed to include o-dianisidine, p-toluidine, 2.2'-azino-di-(3-ethyl-benzothiazoline-o-sulphonic acid) (ABTS), p-diphenylamine sulfonic acid, o-tolidine, natural red (cert.), janus green B (cert.), 2,6-dibromoindophenol sodium salt (prac.) and NN-dimethyl indoaniline (pract.). Suitable titrants are disclosed to include reductants such as gentisic acid, ascorbic acid, hydroquinone, pyrogallol, hydroxylamine, sodium nitrite, sodium bisulfite, sodium thiosulfate, cysteine, hydrazine, ferrous ion and complexes thereof and cuprous ion and complexes thereof.
Despite the existence in the art of tests for the detection of periodontal disease, there remains a need in the art for improved tests with are rapid, inexpensive and sufficiently simple that they may be used with a minimum of difficulty in a dentist's office or by a layperson in the home. Such tests should be eye readable, rather than spectrophotometric, and provide an objective indication of the existence of the disease state with minimal requirements for subjective judgment as to color intensity. Further such tests should avoid the need for adding caustic chemicals such as sodium hydroxide.